LC System Research Articles

B-177 Quantitative Measurement of Vitamin B1 in Whole Blood using a Multiplex ESI-LC-MS/MS System

Abstract Background Vitamin B1 is a water-soluble vitamin required for human health. Thiamine diphosphate (TDP), the active form of vitamin B1, plays an essential role in biochemical pathways required for energy metabolism. TDP is analyzed routinely in the laboratory to assess the nutritional status of post-bariatric surgery patients and other individuals who are at risk of vitamin deficiency. In recent years, demand for vitamin B1 testing has increased dramatically, prompting development of a high throughput assay. Here, we present a method using protein precipitation and multiplex LC-ESI-MS/MS analysis at low pH with reversed-phase parameters that optimize chromatographic separation. The new method was validated for linearity, sensitivity, accuracy, imprecision, specificity, reproducibility, carryover, and stability. Method Whole blood samples were deproteinized using trichloroacetic acid (TCA) after the addition of stable-isotope-labeled TDP (d3-TDP) as an internal standard (IS). TDP was separated by reversed-phase chromatography on Waters XBridge™ Premier BEH C18 columns (2.1 × 100 mm, 2.5 µm) and analyzed on an Agilent StreamSelect™ LC-ESI-MS/MS platform. The StreamSelect™ system consists of four parallel Agilent Infinity Lab LC Series II instruments coupled to a single triple quadrupole mass spectrometer (6470B). TDP was found to interact with metal surfaces in the LC system, so the flow path was passivated using a deactivator solution which improved overall sensitivity and peak quality. TDP eluted in 3.15 ± 0.08 minutes; run times per column for a single stream and four streams were 9 min and 2.25 min, respectively. TDP was analyzed using positive electrospray ionization and multiple reaction monitoring mode with mass transitions of m/z 425.05 > 121.1 (quantifier) and m/z 425.05 > 304.00 (qualifier). Results The method was linear from 20 to 1000 nmol/L with an R2 > 0.99. Replicate analysis of low calibrators to evaluate assay sensitivity demonstrated accuracy within 10% and imprecision of ≤5%. Spike-recovery experiments showed an accuracy of ±15%. TDP measured in purchased, matrix-matched quality control (QC) and expired proficiency testing materials met the specified range for all samples. The intra- and inter-day assay imprecision was ≤3%. The cross-analyte interference study showed the analyte response in the IS-only blank was less than 10% of the LLOQ area. Also, the IS response at the ULOQ was ≤5% of the analyte area. Repeated injections of calibrators and QC materials across the four LC streams showed excellent parity (<2% imprecision). Re-injection reproducibility of extracted samples at 24 and 48 h was within ±2% of time zero concentration. Batch size tests indicated a high degree of robustness required to support the anticipated high testing volumes. Conclusions An accurate, specific, and high-throughput LC-ESI-MS/MS method was developed and validated to measure TDP in whole blood. Simple, quick sample preparation was employed for adaption to a high-throughput platform. StreamSelect™ using four parallel LCs reduced injection times to 2.25 min per sample and significantly minimized mass spectrometer idle time. The assay performance evaluations met our laboratory-developed test acceptance criteria for use in routine clinical laboratory analysis.

B-166 Effect of Urine Adulteration on Quantifying both Delta 8- and Delta 9-carboxy tetrahydrocannabinol using a New UPLC-tandem Mass Spectrometry Method

Abstract Background The legality and use of marijuana is a controversial topic. With increased prevalence of usage, cannabis detection for legal, medical, and workforce drug testing is on the rise. LC-MS/MS quantification of delta 9-carboxy tetrahydrocannabinol (delta 9-THC-COOH) in urine is accepted as the standard to detect marijuana use. One challenge faced by testing laboratories has been identifying adulterated samples. Many products/reagents can be used to manipulate the urine to avoid positive drug testing results. In this study, we developed a dilute-and-shoot strategy to separate and quantify both the delta 8- and 9-THC-COOH in urine samples and tested the effects of adulteration using a combination of nitrite and/or acids which can be used to mask cannabinoid use. Methods Five authentic delta 8- and 9-THC-COOH positive specimens, and one spiked sample were treated with strong acids (1N HCL or 10% TCA), 500 µg/mL nitrite, or a combination. After hydrolysis with sodium hydroxide, the analytes were quantified using a 6495 Mass Spectrometer coupled to a StreamSelect LC system (Agilent). LC separation was performed with a CORTECS T3 column utilizing a shallow gradient. In addition, twelve urine specimens with varying concentrations of delta 8- and 9-THC-COOH were treated with nitrite and screened using a delta 9-THC-COOH immunoassay on the Roche Cobas C501 and analyzed by LC-MS/MS. Results Nitrite did not change the physical appearance or the pH of the urine samples. Strong acid treatment also did not alter the appearance but did drop the urine pH ≤2. Pre-treated specimens with 1N HCL, 10% TCA, or 500 µg/mL of nitrite did not affect the delta 8- and 9-THC-COOH concentrations up to 6 days (refrigerated); however, specimens treated with strong acid combined with 500 µg/mL of nitrite for 30 min made both analytes undetectable. Additional experiments confirmed that 0.5 and 1 mol/L of nitrite lowered the concentration of all analytes, including internal standard, to undetectable levels in 48 h. Twelve additional specimens were collected, 3 with delta 8-THC-COOH, 3 with delta 9-THC-COOH, and 6 with both. All 12 samples screened positive using the Roche immunoassay and were quantified using the LC-MS/MS method. After treatment with 0.5 mol/L of nitrite, 10 of the 12 samples were negative using the immunoassay, but 2 samples remained positive. However, all nitrite treated specimens were negative (<LLOQ) using the LC-MS/MS method and the internal standard was eliminated. Later experiments confirmed that the delta 8- and 9-THC-COOH could be eliminated with as little as 0.1 mol/L nitrite for 30 min. Conclusion The unique dilute-and-shoot method can separate and quantify delta 8- and 9-THC-COOH. In addition, high concentrations of nitrite were shown to degrade both analytes along with the isotopically labeled internal standard. As a result, labs can distinguish true negative delta 8- and 9-THC-COOH (no analyte peaks) from nitrite adulterated specimens since the adulterated samples have no peaks for both the analytes and internal standard. A limitation of the study is that only sodium nitrite was tested. Other adulterants, such as potassium nitrite, pyridinium chlorochromate, peroxide etc. require further investigation.

B-182 Advantage of Core-Shell Particle Morphology Over Fully Porous HPLC Column for the Analysis of Vitamin K Compounds by LC-MS/MS

Abstract Background Vitamin K’s are a group of structurally similar, fat-soluble compounds. There are two natural forms K1 (Phylloquinone) and K2 (Menaquinone), where the later has short and longer chain homologues called Vitamin K2-MK-4 and Vitamin K2-MK-7 respectively. Due to the extremely hydrophobic nature and poor ionization characteristics of these compounds, LC-MS/MS analysis at low concentrations becomes challenging. In this communication we make parallel comparisons of three HPLC columns packed with fully porous and core-shell particles to showcase the superiority of core-shell columns for sensitive detection of vitamin K analytes by LC-MS/MS. Method We compared fully porous (Gemini and Luna, Phenomenex, Torrance, CA) and core-shell (Kinetex EVO, Phenomenex) C18 columns for this study. All columns evaluated were 50 × 2.1 mm with a 5 μm particle size to maintain low back pressure for the viscous organic modifier utilized. Deionized water and a combination of isopropanol and acetonitrile (1:1; v/v) were used as mobile phase A and B respectively for chromatographic separation. An Agilent 1290 Infinity series LC system was employed with a SCIEX 6500 QTRAP mass spectrometer under APCI for MS detection. Analyst software 1.7.2 used for data analysis. Results The LC-MS/MS method had a 4-minute runtime for chromatographic separation of the analytes including 1.5 min of equilibration time. The comparison data revealed the Gemini C18 column shows some higher selectivity and resolution values, whereas both EVO and Luna C18 columns were somewhat comparable. However, the core-shell EVO C18 column displayed much higher efficiency with narrower and compressed peak widths, which dramatically increased the peak height for all 3 analytes exhibiting the highest signal/noise ratio. Conclusion The core-shell Kinetex 5 μm, EVO C18 column with its unique organo-silica grafting shows much higher efficiency and sensitive detection capability for vitamin K analysis by LC-MS/MS compared to that of conventional fully porous silica particles.

St. Nersess Theological Review uses the following Transliteration Table Based on the Library of Congress System

St. Nersess Theological Review uses the following Transliteration Table Based on the Library of Congress System

Quantification of drug metabolising enzymes and transporter proteins in the paediatric duodenum via LC-MS/MS proteomics using a QconCAT technique

Characterising the small intestine absorptive membrane is essential to enable prediction of the systemic exposure of oral formulations. In particular, the ontogeny of key intestinal Drug Metabolising Enzymes and Transporter (DMET) proteins involved in drug disposition needs to be elucidated to allow for accurate prediction of the PK profile of drugs in the paediatric cohort.Using pinch biopsies from the paediatric duodenum (n = 36; aged 11 months to 15 years), the abundance of 21 DMET proteins and two enterocyte markers were quantified via LC-MS/MS. An established LCMS nanoflow method was translated to enable analysis on a microflow LC system, and a new stable-isotope-labelled QconCAT standard developed to enable quantification of these proteins. Villin-1 was used to standardise abundancy values. The observed abundancies and ontogeny profiles, agreed with adult LC-MS/MS-based data, and historic paediatric data obtained via western blotting. A linear trend with age was observed for duodenal CYP3A4 and CES2 only. As this work quantified peptides on a pinch biopsy coupled with a microflow method, future studies using a wider population range are very feasible. Furthermore, this DMET ontogeny data can be used to inform paediatric PBPK modelling and to enhance the understanding of oral drug absorption and gut bioavailability in paediatric populations.

LC Headings and Classification from January – May 2023 Lists

LC Headings and Classification from January – May 2023 Lists

Scout triggered multiple reaction monitoring mass spectrometry for the rapid transfer of large multiplexed targeted methods in metabolomics

The field of metabolomics based on mass spectrometry has grown considerably in recent years due to the need to detect and, above all, quantify a very large number of metabolites, simultaneously. Up to now, targeted multiplexed analysis on complex samples by Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS) has relied almost exclusively on compound detection based on absolute retention times, as in the Scheduled-MRM (sMRM) approach. Those methods turn out to be poorly transferable from one instrument to another and result in a time-consuming and tedious method development involving a significant number of critical parameters that need specific re-optimisation. To address this challenge, we introduce a novel acquisition mode called scout-triggered MRM (stMRM). In stMRM, a marker transition is used to trigger MS analysis for a group of dependent target analytes. These marker transitions are strategically distributed throughout the chromatographic run, and the dependent analytes are associated based on their retention times. The result is a targeted assay that remains robust even in the presence of retention time shifts. A 3 to 5-fold increase in the number of detected transitions associated to plasma metabolites was obtained when transferring from a direct application of a published sMRM to a stMRM method. This significant improvement highlights the universal applicability of the stMRM method, as it can be implemented on any LC system without the need for extensive method development.We subsequently illustrate the robustness of stMRM in modified chromatographic elution conditions. Despite a large change in metabolite’s selectivity, the multiplexed assay successfully recovered 70% of the monitored transitions when consequently modifying the gradient method. These findings demonstrate the versatility and adaptability of stMRM, opening new avenues for the development of highly multiplexed LC-MS/MS methods in metabolomics. These methods are characterized by their analytical transparency and straightforward implementation using existing literature data.

Ultralow flow liquid chromatography and related approaches: A focus on recent bioanalytical applications.

Ultralow flow LC employs ultra-narrow bore columns and mid-range pL/min to low nL/min flow rates (i.e., ≤20 nL/min). The separation columns that are used under these conditions are typically 2-30μm in inner diameter. Ultralow flow LC systems allow for exceptionally high sensitivity and frequently high resolution. There has been an increasing interest in the analysis of scarce biological samples, for example, circulating tumor cells, extracellular vesicles, organelles, and single cells, and ultralow flow LC was efficiently applied to such samples. Hence, advances towards dedicated ultralow flow LC instrumentation, technical approaches, and higher throughput (e.g., tens-to-hundreds of single cells analyzed per day) were recently made. Here, we review the types of ultralow flow LC technology, followed by a discussion of selected representative ultralow flow LC applications, focusing on the progress made in bioanalysis of amount-limited samples during the last 10 years. We also discuss several recently reported high-sensitivity applications utilizing flow rates up to 100 nL/min, which are below commonly used nanoLC flow rates. Finally, we discuss the path forward for future developments of ultralow flow LC.

Molecular scale insights from NMR studies of hybrid systems formed via doping silver QDs in 6CHBT liquid crystal: a quantitative investigation of their optoelectronic properties

ABSTRACT We have successfully synthesised silver quantum dots (Ag QDs) via chemical method. We have studied Ag QDs doped 4-(trans-4-n hexylcyclohexyl) isothiocyanatobenzoate (6CHBT) LC hybrid system and their associated structural and optoelectronic properties using both experimental and theoretical methods. The QDs with sizes 4.27 nm−9.56 nm self-assemble to form nano-micro scale hierarchical structures. Theoretical NMR study has been performed using three different methods HF, B3LYP, and wB97XD for pure and Ag NPs doped LC system. NMR data greatly helps us to explore the phenomenological description of the hybrid system by giving us molecular scale insights. Optical microscope, RAMAN, UV, FTIR, HR-XRD, FESEM-EDX, and Fluorescence data confirm the structural and physio-chemical properties of QDs, LC molecules and as well as their hybrid system. We use a nanoscale predictive model to estimate the effective size to which the bandgap value of Ag QDs in a hybrid system corresponds. The bandgap value of pure Ag QDs is found to be 5.32 eV, whereas upon doping these Ag QDs in LC media, their bandgap falls to a value of 4.08 eV. The estimated size (i.e. corresponding to effective confinement) using a hexahedral or octahedral-shaped particle model comes out to be ~15 nm.

Development of Wall-Coated Open Tubular Columns and Their Application to Nano Liquid Chromatography Coupled to Tandem Mass Spectrometry.

This work presents a study on the application of wall open tubular column (WCOT) in liquid chromatography coupled with tandem mass spectrometry. Each process step reports the column preparation method in detail, subdivided into column pretreatment, silanization, stationary phase coating, and immobilization. Then, an evaluation of the parameters that can affect the efficiency of these columns was made. Atrazine, clomazone, and metolachlor were used as probes during this step. Factors such as stationary phase composition, length, internal diameter, stationary phase mass employed, and injection volume were investigated. In addition, with the help of Knox and Poppe graphs, the columns' performance was evaluated to determine the optimal flow rate and the speed-efficiency relationship, respectively. Based on the results, the best configurations for the WCOT column application to the LC system were defined: length-8 m; inner diameter-25 μm; mass of OV-210-2.5% m/v; and, injection volume-100 nL. Finally, the optimized WCOT column developed in this work was coupled with a commercially-packed trapping column in the nano liquid chromatography system (nanoLC). In this configuration, more significant results were obtained regarding separation resolution, with Rs = 5.9 achieved for the most retained pair of analytes (clomazone and metolachlor).

Women’s Words and the Words of Women in the Oxford English Dictionary

Abstract This article explores datasets curated from the citation evidence in successive editions and revisions of the Oxford English Dictionary (1884–2022), which have been annotated to reflect the gender of the authors and other bibliographical metadata. This exploration aims both to supplement the historical account of the dictionary’s uses of female-authored quotation sources, correcting and elaborating some figures which have previously been reported, and to provide a contemporary account of women’s representation in OED Online, using the revision published in June 2022. In seeking to establish a more objective and empirical basis for judging ‘representativeness’, I treat the OED both as a self-contained bibliographical and lexicographical work, and comparatively, against other comprehensive or very large bibliographical corpora, namely the Garside et al. surveys of early English novels, the Library of Congress Catalog, and the HathiTrust Digital Library. The OED data studied here represents a significant (if restricted) subset, rather than a representative sample, of the OED corpus as a whole: modern (post-1700) quotations from books appearing with their author’s name in the OED evidence are considered. While this approach does not claim to make an objectively complete tally of every woman-authored quotation collected in the OED, it does enable a more detailed and accurate account than has previously been possible, and allows for a number of consistent cross-comparisons. A companion document of Supplementary Data & Notes, available at The Review of English Studies online, describes in technical terms how the data was compiled and the processes and principles by which it was annotated.

The Development of Learning Achievement by Peer-assisted Learning Associated of Students in the Library and Information Science Curriculum

The purposes of this research were the development of learning achievement by peer-assisted learning associated of students in the library and information science curriculum, and to the study satisfaction of students with peer-assisted learning associated in library of congress classification. The population for this research were 19 students of the library and information science curriculum. The instruments for this research were learning achievement test, and satisfaction from. The statistics for data analysis were the percentage, mean, standard deviation, and t-test Independent. The research finds that the development of learning achievement by peer-assisted learning associated of students after learning management has improved meaningfully with the significant level at .05, and the total level of the satisfaction of students with peer-assisted learning associated was high.

A QbD-led simple and sensitive RP-UHPLC method for simultaneous determination of moxifloxacin, voriconazole, and pirfenidone: An application to pharmaceutical analysis.

A novel, quick and precise RP-UHPLC analytical method for the simultaneous determination of moxifloxacin (MFX), voriconazole (VCZ) and pirfenidone (PIR) was developed and validated according to the International Conference on Harmonization guidelines using a QbD-driven response surface Box-Behnken design. The developed method was validated considering the selectivity, sensitivity, linearity, accuracy-precision, robustness, stability, limit of detection and limit of quantification, respectively. Resolution between MFX, VCZ and PIR was achieved using a gradient elution protocol against a Waters Symmetry Shield C18 column (150 × 4.6mm2 , 5μm) using an Agilent 1290, Infinity II series LC system. The method was applied to quantitatively estimate proprietary and in-house prepared pharmaceutical topical ophthalmic formulations containing MFX, VCZ and PIR at wavelength(λmax ) of 296, 260 and 316 nm. The method is sensitive enough to detect up to 0.1ppm of analytes in the formulation. The method was further exploited to study and identify the possible degradation products of the analytes. The proposed chromatographic method is simple, economical, reliable and reproducible. In conclusion, the developed method could be applicable for routine quality control analysis of single or combined MFX, VCZ and PIR-containing units or bulk dosage forms in pharmaceutical industries and research organizations working on drug discovery and development.

Column selection considerations in compact capillary liquid chromatography

Recent years have seen significant advances in compact, portable capillary LC instrumentation. This study explores the performances of several commercially available columns within the pressure and flow limits of both the columns and one of these compact LC instruments. The commercially available compact capillary LC system with UV-absorbance detector used in this study is typically operated using columns in the 0.15–0.3 mm internal diameter (i.d.) range. Efficiency measurements (i.e., theoretical plates, N) for six columns with i.d.s in this range and of varying lengths and pressure limits, packed with stationary phases of different particle diameters and morphologies, were made using a mixture of standard alkylphenones. Kinetic plot comparisons between columns that vary by one (or more) of these parameters are described, along with calculated kinetic performance and Knox-Saleem limits. These theoretical performance descriptions provide insight into optimal operating conditions when using capillary LC systems. Based on kinetic plot evaluation of available capillary columns in the 0.2–0.3 mm i.d. range with a conservative upper pressure limit of 330 bar packed with superficially porous particles, a 25 cm column could generate ∼47,000 plates in 7.85 min when operated at 2.4 µL/min. For comparison, more robust 0.3 mm i.d. columns (packed with fully porous particles) that can be operated at higher pressures than can be provided by the pumping system (conservative pump upper pressure limit of 570 bar), a ∼20 cm column could generate nearly 40,000 plates in 5.9 min if operated at 6 µL/min. Across all capillary LC columns measured, higher pressure limits and shorter columns can provide the best throughput when considering both speed and efficiency.

Effect of Phenoxyethanol and Sodium benzoate on the liquid crystal phase behavior of mixed surfactant system

Surfactants in a mixed concentrated (∼30 wt% and higher)-surfactant system self-assemble to form a variety of liquid crystal (lc) types depending the lc curvature determined by the surfactant chemistries and their relative compositions. Preservatives, fragrances and other minor additives present in an industrial formulation can either incorporate into the lc and/or influence the aqueous phase ionic strength to alter the spatial arrangements of the surfactants and the lc curvature. These mechanisms could additively or competitively determine the lc curvature and thereby the lc types formed. We report on the mechanisms for preservatives Phenoxyethanol (PE) and Sodium benzoate (NaB) governing the lc phase behavior for a mixed concentrated-surfactant system: 16 wt% zwitterionic Cocoamidopropyl betaine (CAPB), and 7 wt% each of anionic Sodium Lauroyl isethionate (SLI), Sodium Methyl Lauroyl Taurate (SMLT), and 6 wt% Lauric acid. The lc type(s), the lc dimensions including inter-lc distances were derived from SAXS and polarized microscopy images. The predominant lc phase without preservatives was H1lc, while anionic SLI and SLMT present in excess were insoluble to form dispersed crystals. The addition of PE molecules resulted in their incorporation in the lc surfactant film to cause a transition to micelles, and Lα lc due to incorporation-induced flattening of the film. NaB molecules prevented micelles formation and preserved the H1lc by serving as a hydrotropic agent. The NOESY spectra for the lc systems with PE or NaB at 0–3 wt% confirmed incorporation of PE in surfactant film as opposed to NaB. For both PE and NaB present in a lc system, the dominance of hydrotropic nature of NaB causing film bending rather than the film flattening effect of PE at low (∼1 wt%) PE levels resulted in H1lc as the predominant phase, and reduced dominance of film bending effect at a high PE level (≥ 2 wt%) led to the formation of Lα lc.

HyperSCP: Combining Isotopic and Isobaric Labeling for Higher Throughput Single-Cell Proteomics.

Recent developments in mass spectrometry-based single-cell proteomics (SCP) have resulted in dramatically improved sensitivity, yet the relatively low measurement throughput remains a limitation. Isobaric and isotopic labeling methods have been separately applied to SCP to increase throughput through multiplexing. Here we combined both forms of labeling to achieve multiplicative scaling for higher throughput. Two-plex stable isotope labeling of amino acids in cell culture (SILAC) and isobaric tandem mass tag (TMT) labeling enabled up to 28 single cells to be analyzed in a single liquid chromatography-mass spectrometry (LC-MS) analysis, in addition to carrier, reference, and negative control channels. A custom nested nanowell chip was used for nanoliter sample processing to minimize sample losses. Using a 145-min total LC-MS cycle time, ∼280 single cells were analyzed per day. This measurement throughput could be increased to ∼700 samples per day with a high-duty-cycle multicolumn LC system producing the same active gradient. The labeling efficiency and achievable proteome coverage were characterized for multiple analysis conditions.

LC Headings and Classification from October 2022 – February 2023 Lists

LC Headings and Classification from October 2022 – February 2023 Lists

Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer's Disease Mouse Model.

Suspension TRAPping filter (sTRAP) is an attractive sample preparation method for proteomics studies. The sTRAP protocol uses 5% SDS that maximizes protein solubilization. Proteins are trapped on a borosilicate glass membrane filter, where SDS is subsequently removed from the filter. After trypsin digestion, peptides are analyzed directly by LC-MS. Here, we demonstrated the use of a low-cost plasmid DNA micro-spin column for the sTRAP sample preparation of a dilution series of a synapse-enriched sample with a range of 10-0.3 µg. With 120 ng tryptic peptides loaded onto the Evosep LC system coupled to timsTOF Pro 2 mass spectrometer, we identified 5700 protein groups with 4% coefficient of variation (CoV). Comparing other sample preparation protocols, such as the in-gel digestion and the commercial Protifi S-TRAP with the plasmid DNA micro-spin column, the last is superior in both protein and peptide identification numbers and CoV. We applied sTRAP for the analysis of the hippocampal proteome from the 5xFAD mouse model of Alzheimer's disease and their wildtype littermates, and revealed 121 up- and 54 down-regulated proteins. Protein changes in the mutant mice point to the alteration of processes related to the immune system and Amyloid aggregation, which correlates well with the known major Alzheimer's-disease-related pathology. Data are available via ProteomeXchange with the identifier PXD041045.

Assessment of external calibration, internal standard calibration and quadruple isotope dilution strategies for the determination of acrylamide in wastewater samples after LC-MS/MS quantification

Acrylamide is a possible neurotoxicant and carcinogen compound and its common usage in various industrial processes causes acrylamide contamination in environmental bodies. For this purpose, there is an increasing demand to develop an accurate and selective analytical method for the determination of acrylamide. In the scope of this study, external calibration (EC), internal standard calibration (ISC) and quadruple isotope dilution (ID4) strategies were compared to each other in terms of accuracy and precision. After the evaluation of these methods, liquid chromatography – quadruple isotope dilution – tandem mass spectrometry (LC-ID4-MS/MS) as a highly accurate and precise analytical approach was proposed to quantify acrylamide in wastewater samples. Several parameters for LC system such as sample injection volume, mobile phase type/composition and mobile phase flow rate were elaborately optimized to enhance peak shape and signal to noise ratio (S/N) belonging to the analyte. Under the optimum chromatographic and mass determination conditions, system analytical performance study was performed to determine dynamic range and limit of detection/quantitation (LOD and LOQ). LOD and LOQ values of the proposed LC-MS/MS method for the analyte were calculated as 1.18 and 3.93 µg/kg; respectively. After the system analytical performance study, the spiking experiments in wastewater samples were carried out for the evaluation of accuracy and applicability of LC-MS/MS method. For wastewater samples, percent recovery results with their percent relative standard deviations (%RSD) obtained from EC, ISC and ID4 strategies were 115.8–177.8% (±1.2–11.4%), 94.5–117.7% (0.3–4.7%) and 99.6–100.9% (0.4–1.7%); respectively. These results prove the superiority of ID4 strategy with regards to accuracy and precision of the results.

Abstract 5289: Proteomic profile of breast carcinomas with unbalanced levels of progesterone receptor isoforms

Abstract Preclinical studies indicate that progesterone receptors (PR) play a relevant role in breast carcinogenesis. Even more, a misbalanced expression of PR isoforms A (PRA) and B (PRB) differentially affect breast cancer progression and only tumors with higher levels of PRA than PRB (PRA-H) respond to an antiprogestin treatment. There are controversial results regarding the prognosis of PRA-H luminal tumors compared with those with the opposite ratio (PRB-H tumors) highlighting the necessity to further understand the role of PR isoforms in tumor progression and to develop novel methods to discriminate these tumors without performing western blot studies. Along this line we have previously studied the transcriptome of PRA-H or PRB-H breast cancer samples, thus our aim is to expand these results and compare the proteome profile of PRA-H and PRB-H samples obtained from postmenopausal breast cancer patients. Nuclear (Nuc) and cytosolic (Cyt) protein fractions were obtained from 18 breast cancer samples classified as PRA-H or PRB-H and they were studied by LC-MS/MS (UltiMate 3000 LC system - Q Exactive HF-X mass spectrometer - Thermo). We observed 289 differentially deregulated proteins in Cyt (164 down and 125 up) and 301 in Nuc extracts (131 down and 170 up; logFC > 1, pval < 0.05). Gene set enrichment analysis of the Nuc fractions showed biological processes related to Cell junction organization (pval = 8,87e-05), Collagen formation (pval = 0,0005), and Cell-Cell communication processes (pval = 0,0035) in PRB-H tumors; while in PRA-H tumors, pathways related to Innate Immune System (pval = 8,8e-05), Class I MHC mediated antigen processing & presentation (pval = 8e-05) and Stabilization of p53 (pval = 0,0051) processes were enriched. Similar trends were observed in Cyt fractions. Individual candidates that were overrepresented in both fractions in PRB-H tumors are BAG3 and GH3, related to cell proliferation, and VWF, related to the activation of the Akt pathway. Overrepresented proteins in both fractions of PRA-H tumors are BST2, IFT27 related to invasion, and MIF associated with poor prognosis. As we were interested in looking for differential expressed proteins that could be tested in plasma samples to discriminate both tumor types, using the Vertebrate Secretome Database we focused on those that might be secreted. Noteworthy is CBP1 which was overrepresented in the PRB-H tumors and this was in agreement with the RNA-Seq data and with results obtained using preclinical models, repurposing this protein as one candidate of concern in the search for biomarkers. Other secretory proteins of interest are SFRP2 and SERPIND1, both highly expressed in PRA-H Cyt fractions. In conclusion, this study underscores the biological differences between PRA-H and PRB-H breast carcinomas and provides candidates that deserve to be tested as surrogate markers in plasma to discriminate patients that may benefit from an antiprogestin treatment. Citation Format: Andres M. Elia, Jana Sanchez, Rui Vitorino, Leo Saldain, Paula Martinez Vazquez, Javier Burruchaga, Eunice Spengler, Javier Muñoz, Paola Rojas, Luisa Helguero, Claudia Lanari. Proteomic profile of breast carcinomas with unbalanced levels of progesterone receptor isoforms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5289.